The present invention relates to novel C3A receptor ligands, pharmaceutical compositions containing these compounds and methods of using the present compounds to treat inflammation.
Anaphylatoxins are 74-77 amino acid bioactive fragments of C5, C3 and C4 that are generated in vivo during complement activation. Binding of the anaphylatoxins to specific cell surface receptors initiates and maintains the inflammatory process. The fragments are believed to elicit mast cell and basophil degranulation with release of histamine, cytokines and other inflammatory mediators and induce smooth muscle cell contraction. They are potent inflammatory mediators, inducing cellular degranulation, smooth muscle contraction, arachidonic acid metabolism, cytokine release, cellular chemotaxis. See Gerard, C., and Gerard, N. P. (1994) Annu. Rev. Immunol. 12, 775-808; Hugli, T. E. (1984) Springer Semin. Immunopathol. 7, 193-219; Bitter-Suermann, D. (1988) in The Complement System, Ed. by K. Rother and G. Till, Springer Verlag, Heidelberg 367-395.
The present fragments have been implicated in the pathogenesis of a number of inflammatory diseases. See Vogt, W. (1986) Complement 3, 177-188; Morgan, B. P. (1994) European J Clin Investigation 24, 219-228. Studies have demonstrated the presence of a C3A receptor (C3A-R) on guinea pig platelets, rat mast cells, human neutrophils, eosinophils and platelets (Bitter-Suermann, D. (1988) in The Complement System, Ed. by K. Rother and G. Till, Springer Verlag, Heidelberg 367-395). A single class of high affinity C3A binding sites has been characterized on human neutrophils and differentiated U937 cells (KIos, A., Bank, S., Gietz, C., Bautsch, W., Kxc3x6hl, J., Burg, M., and Kretzschmar, T (1992) Biochemistry 31, 112741-1282). Competition binding and functional desensitization studies are consistent with the presence of a receptor for C3A which is distinct from the C5A-R (Bitter-Suernann, D. (1988) in The Complement System, Ed. by K. Rother and G. Till, Springer Verlag, Heidelberg 367-395; Klos, A., Bank, S., Gietz, C., Bautsch, W., Kxc3x6hl, J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282). However, there is evidence that C3A and C4A may bind to the same receptor as the two anaphylatoxins cross desensitize guinea pig ileal tissue (Hugli, T. E. (1984) Springer Semin. Immunopathol. 7, 193-219; Bitter-Suermann, D. (1988) in The Complement System, Ed. by K Rother and G. Till, Springer Verlag, Heidelberg 367-395), although other investigators using guinea pig macrophages indicate that there may be separate receptors (Murakami, Y., Yamamoto, T., Imamichi, T., Nagasawa, S. (1993) Immunol. Lett. 36, 301-304). Functional activity of the C 3A-R is sensitive to pertussis toxin, consistent with the binding site being composed of a GPCR (Klos, A., Bank, S., Gietz, C., Bautsch, W., kxc3x6hl, J., Burg, M., and Kretzschmar, T. (1992) Biochemistry 31, 11274-11282).
A complete understanding of the role of C3A in the pathogenesis of the inflammatory response has been hampered by the lack of the cloned receptor. The present invention provides methods of using and functional characterization of human C3A receptor. This same receptor was recently independently cloned from an HL-60 library by low-stringency screening with a fMetLeuPhe receptor probe and, lacking functional data, claimed to be an orphan receptor (AZ3B,8). Mouse L cells expressing AZ3B failed to bind and respond to the agonists examined, although C3A was not tested (Roglic, A., Prossnitz, E. R., Cavanagh, S. L., Pan, Z, Zou, A. and Ye, R. D. (1996) Biochimica et Biophysica Acta 1305, 39-43). The present invention discloses compounds that antagonize C3A receptor function.
Clearly, there is a need for factors that mediate inflammation and their roles in dysfunction and disease. There is a need, therefore, for identification and characterization of compounds which antagonize C3A receptor function, and which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
Thus, C3A ligands offer a unique approach towards the pharmacotherapy of immune and inflammatory diseases such as rheumatoid arthritis, Alzheimer""s disease, psoriasis, gout, multiple sclerosis, systemic lupus erythematosus, glomerulonephritis and adult respiratory distress syndrome.
The present invention involves compounds represented by Formula (I) hereinbelow and their use as C3A receptor ligands which are useful in the treatment of a variety of diseases associated with complement activation and or increased levels of anaphylatoxins, including but not limited to rheumatoid arthritis, Alzheimer""s disease, psoriasis, gout, multiple sclerosis, systemic lupus erythematosus, glomerulonephritis and adult respiratory distress syndrome.
The present invention further provides methods for antagonizing C3A receptors in an animal, including humans, which comprises administering to an animal in need of treatment an effective amount of a compound of Formula (1), indicated hereinbelow.
The compounds of the present invention are selected from Formula (I) hereinbelow: 
wherein:
A represents C1-4 alkylene, unsubstituted or optionally substituted by C1-4 alkyl or aryl; or
A forms a 5-8 membered fused aliphatic ring with the adjacent phenyl ring;
m is an integer from 1 to 3;
each R1 is independently selected from the group consisting of halo, C1-4 alkyl, methanesulfonyl, alkoxy, nitrile, dimethylamine, methylenedioxy and CF3; and
R2 is hydrogen or methyl.
Preferably, A represents phenethyl.
Preferably m is 0.
Preferably, R1 represents hydrogen.
Preferably, R2 represents hydrogen.
As used herein, xe2x80x9calkylxe2x80x9d refers to an optionally substituted hydrocarbon group joined together by single carbon-carbon bonds. The alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. Preferably, the group is linear. Preferably, the group is unsubstituted. Preferably, the group is saturated.
As used herein xe2x80x9ccycloalkylxe2x80x9d refers to 3-7 membered carbocyclic rings.
As used herein xe2x80x9cheterocycloalkylxe2x80x9d refers to 4-7 membered heterocyclic rings containing 1 to 2 heteroatoms selected from N, O and S.
As used herein, xe2x80x9carylxe2x80x9d refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. xe2x80x9cArylxe2x80x9d includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. A preferred aryl group is phenyl.
As used herein xe2x80x9cacylxe2x80x9d refers to alkylcarbonyl.
The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds and diastereomers are contemplated to be within the scope of the present invention.
Preferred compounds in the present invention include:
1-Naphthyloxyacetylarginine;
1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;
(2,2-Diphenylethoxy)acetylarginine;
(2,2-Diphenylethoxy)acetyl-Nxcex1-methylarginine;
(3-Chlorobenzyloxy)acetylarginine;
2-Naphthyloxyacetylarginine;
(2,3-Dimethylphenoxy)acetylarginine;
8-(Quinolinyloxy)acetylarginine;
6-(Quinolinyloxy)acetylarginine;
2-(1-Bromonaphthyloxy)acetylarginine;
(4-Benzyloxyphenoxy)acetylarginine; and
2-(6-Methoxynaphthyloxy)acetylarginine.
More preferred compounds of the present invention include:
1-Naphthyloxyacetylarginine;
1-[7-(4-hydroxyphenylmethyl)naphthyloxy]acetylarginine;
(2,2-Diphenylethoxy)acetylarginine; and
2-Naphthyloxyacetylarginine.
The most preferred compounds of the present invention include:
1-Naphthyloxyacetylarginine; and
(2,2-Diphenylethoxy)acetylarginine.
An especially preferred compound of the present invention is (2,2-Diphenylethoxy)acetylarginine.
The present compounds can also be formulated as pharmaceutically acceptable salts and complexes thereof. Pharmaceutically acceptable salts are non-toxic salts in the amounts and concentrations at which they are administered.
Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, anmmonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present.
The present invention provides compounds of Formula (I) above which can be prepared using standard techniques. An overall strategy for preparing preferred compounds described herein can be carried out as described in this section. The examples which follow illustrate the synthesis of specific compounds. Using the protocols described herein as a model, one of ordinary skill in the art can readily produce other compounds of the present invention.
All reagents and solvents were obtained from commercial vendors. Starting materials (e.g., amines and epoxides) were synthesized using standard techniques and procedures. The present invention provides compounds of formula (I) above which can be prepared using standard techniques. An overall strategy for preparing preferred compounds described herein can be carried out as described in this section. The examples which follow illustrate the synthesis of specific compounds. Using the protocols described herein as a model, one of ordinary skill in the art can readily produce other compounds of the present invention.
All reagents and solvents were obtained from commercial vendors. Starting materials were synthesized using standard techniques and procedures.
Aryloxyacetylarginines (eg. 5) can be prepared on solid phase. An appropriately protected arginine derivative such as Fmocarginine(Boc)2 (1) is coupled to chlorotrityl resin with an amine base such as diisopropylamine to give 2. Deprotection, and derivatization with bromoacetic acid yields the intermediate bromoacetamide 3. Reaction of this with arylalcohols under basic conditions such as potassium carbonate or amine bases in DMSO with heating yields the aryloxyacetyl product 4. Deprotection with TFA in the presence of a cation scavenger such as triisopropylsilane, dimethylsulfide, ethanedithiol, anisole, water, or some combination of these yields the cleaved product 5.
Alkyloxy and aryloxy derivatives can be prepared by coupling an appropriately protected arginine derivative such as Fmocarginine(Mtr) (6) to Wang resin. Deprotection and coupling with an alkyloxyacetic acid or aryloxyacetic acid yields the protected resin bound intermediate 7. Finally, deprotection with TFA in the presence of a cation scavenger yields the final product 8. 
With appropriate manipulation and protection of any chemical functionality, synthesis of the remaining compounds of Formula (I) is accomplished by methods analogous to those above and to those described in the Experimental section.
In order to use a compound of Formula (I) or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
The present ligands can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical, transdermal, or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs and concentrated drops.
Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably, in physiologically compatible buffers or solutions, such as saline solution, Hank""s solution, or Ringer""s solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories.
For topical administration, the compounds of the invention can be formulated into ointments, salves, gels, or creams, as is generally known in the art.
The amounts of various calcilytic compounds to be administered can be determined by standard procedures taking into account factors such as the compound IC50, EC50, the biological half-life of the compound, the age, size and weight of the patient, and the disease or disorder associated with the patient. The importance of these and other factors to be considered are known to those of ordinary skill in the art.
Amounts administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds with low oral bioavailability, relatively higher doses will have to be administered.
Preferably the composition is in unit dosage form. For oral application, for example, a tablet, or capsule may be administered, for nasal application, a metered aerosol dose may be administered, for transdermal application, a topical formulation or patch may be administered and for transmucosal delivery, a buccal patch may be administered. In each case, dosing is such that the patient may administer a single dose.
Each dosage unit for oral administration contains suitably from 0.01 to 500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. The daily dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal routes contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(I). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I). The active ingredient may be administered from 1 to 6 times per day, preferably once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.
As used herein, xe2x80x9ctreatmentxe2x80x9d of a disease includes, but is not limited to prevention, retardation and prophylaxis of the disease.
Diseases and disorders which might be treated or prevented, include immune and inflammation-related diseases or disorders such as rheumatoid arthritis, Alzheimer""s disease, psoriasis, gout, multiple sclerosis, systemic lupus erythematosus, glomerulonephritis and adult respiratory distress syndrome.
Composition of Formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogs.
Typical dermal and transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose.
No unacceptable toxological effects are expected when compounds of the present invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated by the tests indicated hereinbelow.
Stable Expression of C3A Receptor in RBL-2H3 Cells
To prepare C3A receptor for expression in mammalian cells, a 1.6 kb cDNA fragment was obtained by PCR amplification that encompassed the entire C3A Receptor open reading frame. This fragment was subcloned into KpnI/Hind III sites of the mammalian expression vector, pCDN (Aiyar, N., et al (1994) Mol. Cell. Bio. 131, 75-86). Oligonucleotide primers used for PCR amplification were 5xe2x80x2-GA AGT GGT ACC ATG GCG TC-3xe2x80x2 and 5xe2x80x2-GC TCC AAG CTT TCA CAC AGT TG-3xe2x80x2 (the translation start and stop codons are underlined). RBL-2H3 cells were electroporated with C3A in the pCDN mammalian expression vector (Aiyar, N., et al (1994) Mol. Cell. Bio. 131, 75-86), exactly as described (DeMartino, J. A., et al (1994) J. Biol. Chem. 269, 14446-14450). Individual G418 resistant (400 xcexcg/ml) colonies were isolated and expanded. Clonal cell lines expressing C3A receptor, as determined by ability of the cell line to respond to C3A in a calcium mobilization assay, were chosen for further functional and binding studies.
Preparation of Membranes
RBL-2H3 cells expressing the human C3A receptor (hC3AR) were cultured to confluency at 37xc2x0 C. in a humidified incubator with 5% CO2/95% air, in Earls MEM supplemented with non-essential amino acids, 10% fetal calf serum and 400 xcexcg/ml G418. Although this cell line is normally adherent, nonadherent cells are always present in cultures. The nonadherent cells were adapted to grow in suspension. Nonadherent cells from three T-150 flasks were centrifuged at 1,000xc3x97g for 10 min and resuspended in 50-ml of the above medium in a 250 ml shake flask and over 7-10 days the cells were expanded to 2.51 in a spinner flask. Cells were harvested by centrifugation, 1,000xc3x97g for 10 min at 4xc2x0 C., and membranes were isolated using a modification of the procedure of Ross et al., (1977). Briefly, the cell pellet was washed with PBS and resuspended in 30 ml of hypotonic membrane buffer (20 mM Tris, pH 7.5, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 xcexcM leupeptin, 1 xcexcM pepstatin A) and incubated on ice for 5 min. The cell suspension was homogenized in 40 ml Dounce homogenizer and centrifuged at 1,000xc3x97g for 15 min to remove nuclei and cellular debris. Cell membranes were pelleted at 100,000xc3x97g for 30 min at 4xc2x0 C. Membranes were resuspended in membrane buffer with 10% sucrose and layered over membrane buffer with 40% sucrose and centrifuged at 100,000xc3x97g for 90 min at 4xc2x0 C. Membranes at the interface were isolated and collected by centrifugation at 100,000xc3x97g for 30 min. The membrane pellet was resuspended in 5.0 ml of membrane buffer and aliquots stored at xe2x88x9280xc2x0 C. Protein concentration was quantified using the BCA protein assay reagent (Pierce, Rockford, Ill.).
Scintillation Proximity Assay
All assays are performed in a 96-well micro-titre plate format. The 96-well plates (1450-401) are obtained from Wallac, Turku, Finland. Human anaphylatoxin C3A was obtained from Advanced Research Technologies, San Diego, Calif. with Bolton-Hunter custom iodination being performed by NEN Research Products, Boston, Mass. with specific activity of 2200 Ci/mmol. Wheatgerm agglutinin SPA (Scintillation Proximity Assay) beads were obtained from Amersham Corp., Arlington Heights, Ill. The binding buffer consists of 20 mM Bis-Trispropane, 25 mM NaCl, 1 mM MgSO4, 0.1 mM EDTA at pH 8.0. Each reaction mixture contains: 125I C3A (25 pM, obtained from NEN, Boston Mass.), wheatgerm agglutinin SPA beads (0.1 mg), 0.35 xcexcgs of RBL-2H3 C3A receptor membranes (this may vary with quality of membrane preparation), 23 ug/ml BSA and 0.03% CHAPS in binding buffer.
The membranes were prebound to SPA beads for 30 minutes on ice while shaking. The mixture of membranes and beads were centrifuged for three minutes at 2000 rpm. The supernatant was removed and the pellet was resuspended to original volume in binding buffer with 50 ug/mL BSA. Samples of interest were dissolved in neat DMSO to yield a 20xc3x97 solution followed by a 1:1 mixture with H2O to yield a 10xc3x97, 50% DMSO working solution. The order of addition was 10 uLs sample, 45 uLs membrane bound SPA beads followed by 45 uLs of radiolabled ligand in binding buffer containing 0.06% CHAPS. The plates were covered with plate sealers from Dynex Technologies, Inc, and shaken for 20 minutes and incubated an additional 40 minutes at room temperature. The plates were then centrifuged for three minutes at 2000 rpm followed by counting on the Wallac 1450 Micro Beta Plus Liquid Scintillation counter.
Calcium Functional Assays
7TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day  greater than 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Agonists presenting a calcium transient are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
Calcium Mobilization: C3a-induced Response in RBL-2H3 cells carrying C3a receptor:
Bioassays:
The functional activity of an antagonist of the C3a receptor is demonstrated using the C3a-induced Ca2+ mobilization in RBL-2H3 cells stably expressing C3a (RBL-2H3-C3a).
RBL-2H3-C3a Cell Culture Conditions:
RBL-2H3-C3a cells were cultured to near confluence in T-150 flasks at 37xc2x0C. in a humidified incubator with 5% CO2/95% air in Earls MEM with Earls salts (Gibco) supplemented with non-essential amino acids and L-glutamate, with 10% fetal calf serum (Gibco) and 400 ug/ml G418 (Gibco).
Fluorescent Measurements-Calcium Mobilization:
The functional assay used to assess antagonist activity of compounds was C3a-induced calcium mobilization in intact RBL-2H3-C3a cells. Cells were washed with 50 mM Tris, pH 7.4 containing 1 mM EDTA. The [Ca2+]i was estimated with the calcium fluorescent probe-fura 2 (Grynkiewicz, et al., J. Biol. Chem., 1985, 260, 3440-3450). The media was aspirated from RBL-2H3-C3a cells that were near confluence in T-150 flasks then 40 ml in Krebs Ringer Hensilet containing 0.1% BSA, 1.1 mM MgCl2 and 5 mM HEPES, pH 7.4 (buffer A) was added. The diacetoxymethoxy ester of fura 2 (fura 2/AM) was added at a concentration of 2 xcexcM and incubated for 45 min at 37xc2x0 C. Buffer A was aspirated off the RBL-2H3-C3a cells and 40 ml of Buffer A was added to the cells and incubated for an additional 20 min to allow complete hydrolysis of the entrapped ester. Buffer A was aspirated and cells covered with xcx9c5 ml of Delbeccos Phosphate Buffered Saline with 1 mM EDTA (no calcium or magnesium) for 5 min at 37xc2x0 C. Buffer is aspirated off and 40 ml of buffer A added to the cells which were then mechanically detached from the flasks. RBL-2H3-C3a cells were maintained at room temperature until used in the fluorescent assay which was performed within 3 hours.
The fluorescence of fura 2 containing cells was measured with a fluorometer designed by the Johnson Foundation Biomedical Instrumentation Group. The fluorometer was equipped with a temperature control and a magnetic stirrer under the cuvette holder. Wavelengths were set at 340 nm (10 nm band width) for excitation and 510 nm (20 nm band width) for emission. All experiments were performed at 37xc2x0 C. with constant stirring. For compound studies, fura 2 loaded cells were centrifuged and resuspended in buffer A containing 1 mM CaCl2 minus BSA at 106 cells/mL. For assessment of agonist activity, a 2 mL aliquot of RBL-2H3-C3a cells was added to a cuvette and warmed in a water bath to 37xc2x0 C. The 1 cm2 cuvette was transferred to the fluorometer and fluorescence was recorded for 15 seconds to ensure a stable baseline before addition of compound. Fluorescence was recorded continuously for up to 2 mins after addition of compounds to monitor for the presence of any agonist activity.
For antagonist studies, varying concentrations of compounds or vehicle were added to the fura 2 loaded RBL-2H3-C3a cells and monitored for 1 min to ensure that there was no change in baseline fluorescence followed by the addition of 1 nM C3a. The maximal [Ca2+]/fura 2 fluorescence was then determined for each sample. The [Ca2+]i was calculated using the following formula:             [              Ca                  2          +                    ]        i    =      224    ⁢          xe2x80x83        ⁢          (      nM      )        ⁢                  F        -                  F          min                                      F          max                -        F            
The percent of maximal C3a (1 nM) induced [Ca2+]i was determined for each concentration of compound and the IC50 defined as the concentration of test compound that inhibits 50% of the maximal C3a response. Concentration response curves (5-7 concentrations) were run.
High-Throughput-Screening-Calcium Assay:
The calcium assay described above was converted to a high-throughput-screen (HTS) with the use of a 96 well Fluorescent Imaging Plate Reader (FLIPR) from Biomolecular Devices. This technology allows the measurement of the intracellular calcium mobilization in cells attached to the bottom of a 96 well plate.
For this procedure, cells were obtained from the T-150 flasks as described above. The cells were plated into the 96 well plate at 30,000 cells/well. With incubation in a humidified environment in a cell incubator at 37xc2x0 C. for 18-24 hours, the cells attached to the bottom surface of the 96 well plate.
The FLIPR works best with the visible wavelength calcium indicators, Fluo-3 and Calcium green-1. Both of these dyes have been used successfully for the HTS assay, but Fluo-3 was generally used. Typically 4 uM Fluo-3 was loaded into the cells for 1 hr at 37xc2x0 C. in cell media without fetal calf serum and with 1.5 mM sulfinpyrazone to inhibit dye release from the cells. The media is aspirated from the cells and fresh media was added for 10 min at 37xc2x0 C. to allow hydrolysis of the dye and remove extracellular dye. The media was aspirated and replaced with KRH buffer (buffer A above). After 10 min at 37xc2x0 C. the cells were placed in FLIPR apparatus for analysis.
FLIPR has 3-96 well plates. In addition to the plate with dye loaded cells, there is a plate containing varying concentrations of compound or vehicle and the third plate has the agonist at varying concentrations to establish agonist potency or a single concentration, e.g., 1 nM of C3a for antagonist activity. For antagonist studies, FLIPR obtains a baseline fluorescence for xcx9c30 sec, then it adds the compounds to all 96 wells simultaneously and begins to monitor changes in intracellular Ca2+. After 2 min, the contents or the agonist plate is added to the cells. The maximal Ca2+ response (in optical units) for 1 nM C3a in the presence of vehicle (100%) or the various concentrations of compound is determined. Inhibition curves were generated essentially as described for the single cuvette Fura-2 assay described above.